Process for preparing improved acth



United States Patent rnocnss FOR PREPARING IMPROVED ACTH No Drawing.Application November 14, 1956 Serial No. 621,984 I 15 Claims. or. 16774) This invention is concerned generally with purified extracts ofpituitary glands having the activity of the adrenocorticotropic hormone.More particularly, it relates to purified hydrolysates of theadrenocorticotropic hormone which possess the property of producingremission of clinical and laboratory symptoms of reheumatoid arthritisand related diseases, and which exhibit to a lesser extent or not at allthe undersirable side effects produced by the adrenocorticotropichormone itself.

This application is a continuation-in-part of our application Serial No.225,674, filed May 10, 1951, which, in turn, is a continuation-impart ofapplication Serial No. 166,546, filed June 6, 1950, both now abandoned.

The adrenocorticotropic hormone, hereinafter referred to in thisapplication by the abbreviated term ACTH, can be isolated, insubstantially pure form, from the pituitary glands of sheep according tothe procedure described by Li et al. (J. Biol. Chem. 149, 413-424(1943)); or from the pituitary glands of hogs by the method of Sayers etal. (J. Biol. Chem. 149, 425, (1943)). This hormone has been reported byvarious Workers to produce remission of the clinical symptoms ofreheumatoid arthritis. When ACTH is used in clinical treatment, however,it has been found to cause undesirable side effects, the most importantof which is water retention (antidiuretic effect) which results in edemaand weight gain, heart strain and occasionally congestive heart failure.Other undesirable side effects include occasional hypertension, a risein the uric acid-creatinine ratio, sodium retention, and disappearanceof eosinophiles from the blood.

The adrenocorticotropic hormone, as well as crude extracts thereof, hasbeen subjected to hydrolytic cleavage by prior workers, and thehydrolysates have been tested for hormonal activity. Although some ofthese prior hydrolysates have been found to possess adrenocorticotropicactivity, none has been found to be free of the undesirable side effectscharacteristic of ACTH itself. For example, when ACTH is hydrolyzed bymeans of acids, or by the enzyme pepsin as described in the prior art,the resulting ACTH hydrolysates (although possessing clinical activityin the treatment of rheumatoid arthritis and related diseases) haveinvariably shown the undesirable side effects characteristic of ACTH,even where these hydrolysates have been subjected to a purificationoperation. Other methods of hydrolysis, utilizing aqueous alkali, or theenzyme trypsin, have proved entirely unsatisfactory for producing animproved product since the ACTH hydrolysates thus obtained are nearlydevoid of adrenocorticotropic activity.

Moreover, in the case of the active hydrolysates obtained when, ACTH ishydrolyzed by means of acids or pepsin, the ACTH hydrolysates arecontaminated with unwanted substances of lower or no ACTH activity whichappear to be high molecular weight substances of a proteinaceous nature.Various methods have been reported for separating these substancespresent in the hydrolysis mixture from the material showing maximumadrenocorticotropic activity. A moderate degree of purification has beenachieved by prior workers by treating the hydrolysis mixture withtrichloroacetic acid whereby unwanted substances are precipitated fromthe solution. Crude ACTH hydrolysates have also been freed of suchsubstances present in the mixture by subjecting said bydrolysates todialysis treatment. Although these prior methods have accomplished amoderate increase in the adrenocorticotropic activity of the ACTHhydrolysates, they have uniformly failed to give a product free of theundesirable side effects hereinabove set forth. Moreover, these priorpurification methods utilizing trichloroacetic acid or dialysis areinconvenient to carry out, and entirely unsuitable for use inlarge-scale purification operations.

It is now discovered, according to the present invention, that acidhydrolysates of ACTH can be prepared which possess clinical activityequal to or superior to that shown by ACTH, and which, in clinical use,do not exhibit the undesirable side effects uniformly noted with ACTHitself. We have furtherdiscovered that improved acid hydrolysates whichpossess high ACTH activity, but which do not exhibit the undesirableside effects characteristic of ACTH, can be prepared utilizing, asstarting materials, pepsin digests of ACTH and concentrates thereof.This discovery is particularly surprising in view of the fact that suchpepsin digests, although themselves constituting a hydrolyzed form ofACTH, have invariably exhibited, in clinical use, the sodium retentionand antidiuretic effect uniformly noted with ACTH itself.

Moreover, we have discovered that our novel procedure is generallyapplicable for converting ACTH substances, derived from pituitary glandsand characterized as having both adrenocorticotropic and antidiureticactivity, -to ACTH hydrolysates having substantially unimpairedadrenocorticotropic activity and substantially no antidiuretic activity.The ACTH substances which can be utilized as source materials in ourprocess include purified .ACTH, crude concentrates of ACTH, pepsindigests of such ACTH products, and the like, which are obtained from thepituitary glands of hogs, sheep and cattle (beef). The discovery, thatpituitary glands of sheep and cattle serve equally well as sourcematerials for the preparation of ACTH hydrolysates possessing highadrenocorticotropic activity while beingfree of the undesirable sideeffects of ACTH itself, is especially noteworthy since it has commonlybeen belived that hog pituitaries are the most practical source of ACTHactivity.

It has been further discovered that such ACTH hydrolysates can bepurified by novel purification methods which are adapted for large-scaleoperation. Utilizing these purification methods, we have been able toprepare purified ACTH hydrolysates which possess over thirteen times theadrenocorticotropic activity possessed by pure ACTH itself.

In conducting the hydrolysis of ACTH according to our procedure, pureACTH (as prepared by Li et al. (J. Biol. Chem. 149, 413-424 (1943)) orcrude extracts of pituitary glands such as the acid-acetone powder of Liet al. above cited, or the crude prolactin? described by Fishman (J.Biol. Chem. 167, 425 (1947)) are hydrolyzed with a non-oxidizing mineralacid under carefully controlled conditions. In its broadest aspects,this hydrolysis operation is carried out under sufficiently drasticconditions merely to inactivate the components of the ACTH which areresponsible for the undesirable side effects, without, at the same time,inactivating the simultaneously liberated substance or substancesresponsible for the adrenocorticotropic activity. Any non-oxidizingmineral acid, such as hydrochloric acid, hydrobromic acid, sulfuricacid, phosphoric acid, and the like, can be utilized in this hydrolysisoperation. The concentration of the acid can be varied, although for anygiven hydrogen ion concentramust be adjusted to achieve the properdegree of hydrolysis.

It is ordinarily preferred to employ a mineral acid having aconcentration of approximately 0.3 N (which corresponds to a pH of0.523) as the hydrolizing agent, and to carry out the hydrolysis at atemperature near 100 C. for a period of about one hour. The ratio ofmineral acid hydrolizing agentto ACTH should be relatively large so thatactual concentration of the mineral acid in the hydrolysis mixture issubstantially 0.3 N throughout the course of the hydrolysis reaction,said pH being unafiected by the relatively small amount of ACTH presentin the hydrolysis solution at the start of the treatment, or by theACTH-active material and the hydrolysis products present in thehydrolysis solution at the end of the hydrolysis period. The conditionsof acid concentration, temperature, and time of hydrolysis can be variedonly within certain limits. If these conditions are too mild (too lowacid concentration, too short a time, too low a temperature) the productretains its undesirable side effects; if the conditions are too severe,the product shows a partial or complete loss of ACTH activity. Forexample, when ACTH is hydrolyzed in 0.1 N hydrochloric acid at 100 C.for one hour, according to the prior art procedure described by Li,Evans and Simpson (J. Biol. Chem. 149, 413 (1943)), or using stillweaker acid i. e. 0.1 N to 0.0000001 N, corresponding to a pH of 1.0 to7.0, on the boiling water bath for one to three hours, as disclosed byCollip (Symposia Quant. Biol. 5, 212), the ACTH activity is retained,but the product possesses to a marked extent the undesirableantidiuretic effect. Similarly, when the hydrolysis is carried out in 6N hydrochloric acid at 37 C. for four hours (cf. Li, Macy Foundation,Transactions of the Seventeenth Meeting, Conference on Metabolic Aspectsof Convalescence, New York, N. Y., 1948, p. 114), the product causes aretention of sodium in clinical trials. Increase in time of hydrolysis,utilizing 6 N acid at 37 C., leads to a loss of ACTH activity.

For the preparation of an ACTH product having substantially unimpairedadrenocorticotropic activity and substantially no antidiuretic activity,the following optimum conditions have been established: For hydrolysisfor one hour at 100 C., the acid concentration'in the hydrolysismixture, throughout the course of the hydrolysis reaction,

must be greater than 0.1 N, but not greater than 0.6 N. Thus, theconcentration of mineral acid in the hydrolysis mixture should be withinthe range of 0.2 N to 0.6 N, and the pH of the mixture should thereforebe between pH 0.22 and pH 0.7; the pH may either be determined by directmeasurement of the hydrolysis mixture or may be calculated from thenormality, N, by means of the following equation: pH=log N. When 0.3 Nacid (pH 0.523) at 100 C. is used the hydrolysis must be carried out formore than fifteen minutes, but for less than four hours. For hydrolysisin 0.3 Nacid for one hour, the temperature must be greater than 60 C.,but less than 115 C. Thus, it is ordinarily preferred to hydrolyze theACTH substance with an aqueous mineral acid having a normality betweenabout 0.2 N and 0.6 N (pH 0.22 to pH 0.7), at a temperature betweenabout 60 and 115 C., and for a period of time between approximatelythirty minutes and one hour. When utilizing hydrolysis conditions withinthe range: 0. 2N acid (pH 0.7) at about 100115 C. to 0.6 N acid (pH0.22) at about 60-80 C.,the time of heating can be increased up to twohours, if desired, whereas, utilizing hydrolysis conditions within therange: 0.3 N acid (pH 0.523) at about 100-115 C. to 0.6 N (pH 0.22) acidat about 80-100 C., the time of heating can be decreased to aboutfifteen minutes. It is also evident that higher acid concentrations thanthose preferred can be employedif the time of heating is shortened orthe temperature lowered. Correspondingly, the other variables of timeand. temperature 4 can be altered within reasonable limits to achievethe desired result of a product which possesses substantially unimpairedACTH activity and which is free from the unwanted side effects ofantidiuretic action and sodium retention.

We have discovered that the ACTH hydrolysates thus obtained can bepurified so as to produce purified ACTH hydrolysates free of the sideeffects characteristic of ACTH and possessing over 1300% of theadrenocorticotropic activity of ACTH. In conducting this purificationoperation, we ordinarily employ one or more of several purificationoperations.

It has been found that a considerable degree of purification of the ACTHhydrolysate is accomplished by extracting an aqueous solution of saidACTH hydrolysate with a mixture of phenol and an organic solvent whichis either partially or totally immiscible with water such as butanol,chloroform, and the like. In carrying out this extraction operation, itis ordinarily preferred to dissolve the ACTH hydrolysate (which can bereadily obtained in solid form by drying the hydrolysis mixture in thefrozen state) in waterand washing the aqueous solution with a mixtureconsisting of phenol and organic solvent. When chloroform is employed, aratio of two parts phenol 'to one of chloroform is preferred, althoughthe ratio of phenol to chloroform can be varied if desired. The aqueoussolution is then washed with the organic solvent (chloroform, butanoland the like) and then with ether to remove phenol, and the resultingaqueous solution is dried from the frozen state to produce the purifiedACTH hydrolysate in solid form.

The foregoing extraction procedure has been found to remove unwantedsubstances, described above, from the ACTH hydrolysate. Thepurified ACTHhydrolysate thus produced, when tested according to the rat assay ofSayers et a1. (Endocrinology, 42, 378 (1948)), gives a value ofapproximately which is equivalent to the value obtained for ArmourStandard ACTH. When administered to a patient with rheumatoid arthritis,this product is fully active in inducing remission of the symptoms, theeflect being equivalent to the clinical responses obtained with ArmourStandard ACTH (L. A. 1050). Moreover, in clinical use, this product issubstantially free of side effects: the'sodium retention, waterretention and eosinophile drop in the patient being negligible ascompared with the values consistently observed in similar treatment withACTH.

The purified ACTH hydrolysate prepared utilizing the phenol-organicsolvent treatment described above can be further purified if desired.This further purification is accomplished by dissolving the product inmethanol and diluting the methanol solution with ether, whereupon a morepurified ACTH hydrolysate precipitates and can be recovered byfiltration. This latter product is slightly more active than thematerials obtained following the phenol-organic solvent treatment, ismore stable, and is substantially free of hydrochloric acid which isremoved by this procedure. This methanol-ether precipitated ACTHhydrolysate is also less hygroscopic, denser, and is in generallyimproved physical state as compared with the starting material.

Where it is desired to prepare a highly-active product which is free ofside effects, the crude ACTH hydrolysate is first subjected to thephenol-organic solvent extraction procedure followed by precipitation ofthe partially purified hydrolysate'from methanol solution by theaddition of ether. This purified ACTH hydrolysate fraction is thendissolved in methanol and the resulting solution is treated with amethanol solution containing triethylamine, at which time a whiteprecipitate forms. This product, which is a still more purified form ofthe ACTH hydrolysate, in the form of the free base, is readily recoveredby centrifugation or filtration. It is highly active, showing an assayactivity of 200% (according to the rat assay referred to hereinabove),and canbe used for 5 the clinical treatment of rheumatoid arthritiswhile at the same time producing none of the undesirable side effectscharacteristic of the ACTH.

Alternatively, the ACTH hydrolysate can first be converted to the freebase by treatment with triethylamine in methanol solution. The resultingprecipitate of ACTH-free base can be dissolved in Water and purified bythe phenol-solvent washing, and the phenol extraction procedure. Thus,the purification involves extracting an aqueous solution of the freebase form of a crude ACTH hydrolysate with a mixture of phenol andanother organic solvent, thereby extracting unwanted substances andleaving the base form of the active portion of the ACTH hydrolysate inthe aqueous layer; this is followed by'extraction of said free base fromthe aqueous solution by means of phenol. The ACTH hydrolysate inpurified form is then recovered by diluting the phenol solution withether and extracting with acidified water.

i In its broadest aspects the hereindisclosed purification procedurecomprises the purification of an ACTH hydrolysate, said hydrolysatebeing obtained by hydrolyzing ACTH with aqueous mineral acid, by one ormore of sev- 6 at the temperature and for the period of time indicatedin the table. In each case, the hydrolysis solution was evaporated todryness in vacuo, and the dry residue was triturated with ether. Theether-insoluble material was then dissolved in methanol (approximately 1ml.) and precipitated by the addition of ether (about 10 ml.). Theprecipitates were washed with ether and dried.

The ACTH activities of the ACTH hydrolysates thus obtained weredetermined utilizing the rat assay of Sayers et al. (Endocrinology 42,378 (1948)). The antidiuretic activity of each of these ACTHhydrolysates in rats, which has been demonstrated to be a reliablemeasure of the side effects (i. e., water retention and sodiumretention) produced by ACTH products upon clinical administration, wasdetermined as follows: One group of rats was administered, by injection,1.2 mg./kg. of ACTH hydrolysate, and a second group of rats (uninjected)was set aside as controls; a standard dose of water was administered toeach group of rats, and the time required for the animals to excrete 50%of the administered water was measured; the time required for the 50%excretion of the water by the test group, as compared with the timerequired for 50% excretion by the controls, was taken as a measure ofthe antidiuretic activity of the sample.

The hydrolysis conditions, the ACTH activity of the ACTH hydrolysate,the antidiuretic effect and the suitability of the product for clinicaluse are summarized in the following table.

Approxi- Side Effects as Acid Concentration in mate pH Time, Temp., ACTHMeasured by Hydrolysis Solution of Hydrol- Hours 0. Activity,Antidiuretic Product ysis Percent Test Solution 3 0,1 N 1.0 1 100 95 ADPositive-.. Unsatisfactory. 0.3 N 0. 5 1 100 100 AD Negative-Satisfactory. 0.6 N 0. 2 1 100 70 do Do. 1.5 N 1 100 Unsatisfactory. 6 NH 0 4 37 Do. 6 N H 0 8 37 Do. 6 N H 0 16 37 Do. 0.3 N 0. $4 100 Do. 0.3N 0. 5 100 Satisfactory. 0.3 N 0. 5 2 100 D0. 0.3 N 0. 5 4 100 NoneUnsatisfactory. 0.3 N 0.5 1 37 AD Positive Do. 0.3 N 0.5 1 60 ADPositive Doubtful.

slight) 0.3 N 0.5 1 115 75 A N egative Satisfactory. 0.3 N 0.5 1 148None Unsatisfactory. 0.3 N 0.5 1 100 105 AD Negative- Satisfactory. 0.8N 0.5 1 100 110 do Do.

1 Hydrolysis solution diluted with about volumes of water beforeevaporation to dryness. 2 Adjusted to pH 3.9 with Ba(OH)i and filteredbefore evaporated to dryness. 3 pH of hydrolysis solution remainssubstantially constant throughout the hydrolysis reaction.

is substantially free of undesirable side effects characteristic of theACTH and can be utilized as set forth hereinabove. If desired, thisproduct can be subjected to further purification treatment involvingprecipitation of the partially-purified salt of the ACTH hydrolysatefrom methanol solution by the addition of ether, which procedures yieldproducts which have substantially no undesirable side effects inclinical studies. Alternatively, where the free base of the ACTHhydrolysate is desired, instead of the salt thereof, the salt isdissolved in methanol and precipitated therefrom by the addition oftriethylamine to the methanol solution, thereby precipitating the freebase of the ACTH hydrolysate.

The following examples illustrate methods of carrying out the presentinvention, but it is to be understood that these examples are given forpurposes of illustration and not of limitation.

Example 1 Seventeen samples of ACTH (prepared substantially as describedin J. Biol. Chem. 167, 425 (1947)) of about mg. each were dissolved in20 ml. portions of dilute aqueous mineral acid (the particular acid andits concentration utilized for the hydrolysis of each sample is given inthe table hereinbelow) and the resulting solution heated Example 2 Asample of ACTH was prepared according to the method described in J.Biol. Chem. 167, 425 (1947) for the isolation of purified ACTH. It wasfound by the rat assay to possess marked antidiuretic activity. Thus,the time required for an excretion of 50% of an administered dose ofwater was 214 minutes in rats given 1.2 mg./kg. of the ACTH sample,whereas the corresponding time for the control (uninjected) rats was 79minutes. The same ACTH preparation was tested clinically. It exerted. amoderate effect on the patients arthritis, but caused some retention ofsodium and a marked water retention.

Five and one-tenth grams of the ACTH sample of the preceding paragraph,were dissolved in ml. of boiling 0.3 N hydrochloric acid, and thesolution (pH about 0.5) was boiled under reflux for one hour. Thesolution (pH about 0.5 was then cooled and evaporated to dryness invacuo, while maintaining the temperature below room temperature.dissolved in 100 ml. of water and this solution was washed with two 100ml. portions of a mixture (2:1) of liquefied phenol and chloroform. Theaqueous solution was then washed with chloroform and ether to removephenol, and lyophilized. The lyophilized residue was The residualmaterial was dissolved in 20 ml. of methanol and precipitated by theaddition of 115 ml. of ether. The precipitate was dried in vacuo to givea pale brownish-tan powder, 715 mg. In the rat assay, the product gave avalue of 100%, approximately equivalent to the activity of ArmourStandard ACTH. In a clinical trial, it was administered to a patientwith rheumatoid arthritis in a dosage of 12.5 mg. every 6 hours for oneday. It was found to be fully active in inducing remission of symptoms,the effect being equivalent to the clinical response obtained withArmour Standard ACTH (L. A. 1050). No significant sodium or waterretention in the patient was noted. 7

Analyses showed the composition of the product to be: carbon, 38.79%;hydrogen, 6.49%; nitrogen, 14.79%; chlorine, 16.27%; sulfur, 2.07%. Itcontained 3.92% of amino-nitrogen (Van Slyke). A complete acidhydrolysis (6 N hydrochloric acid at 150 C. for 16 hours), followed by aVan Slyke determination, gave a value of 14.59% peptide-nitrogen.

Example 3 A portion of 1.71 g. of an ACTH hydrolysate fraction, whichhad been prepared and purified according to the method indicated inExample 2, was dissolved in 17 ml. of cold C.) methanol and treated witha solution of 1 ml. of triethylamine in 5 ml. of methanol. Afterstanding at 5 C. for five minutes, the white precipitate was removed bycentrifugation. It was washed with four ml. portions of cold (5 C.)methanol, three 40 ml. portions of ether, and dried in vacuo. Theproduct, 436 mg., showed an assay value of 220%.

Example 4 A solution of 20.0 g. of crude prolactin (prepared asdescribed in J. Biol. Chem. 167, 425 1947)) in 400 ml. of 0.3 Nhydrochloric acid was boiled under reflux for one hour. The solution (pHabout 0.5) was cooled and concentrated to dryness in vacuo below roomtemperature. The residue was disolved in 500 ml. of water and theaqueous solution was washed with two 375 ml. portions of 2: 1 (v./v.)liquefied phenol; chloroform. The aqueous solution was then washed withthree 1 liter portions of chloroform and one 1 liter portion of ether,after which it was filtered and lyophilized. The lyophilized materialwas triturated with 78 ml. of methanol, and the portion of the materialinsoluble in the methanol was discarded. The methanolic solution wastreated with 460 ml. of ether, and the resulting precipitate wascollected by centrifugation and dried. The yield at this stage was 2.65g.

The 2.65 g. of material was dissolved in 26 ml. of methanol, a smallinsoluble residue was removed, and the solution was treated with 9 ml.of a 5:1 (v./v.) methanol-triethylamine mixture. formed was collected,washed with four 10 ml. portions of cold methanol and three 40 ml.portions of ether and dried in vacuo. The yield in purified free basewas 606 Example 5 A 30 g. portion of crude ACTH designated as acidacetone powder (prepared from sheep pituitaries substantially accordingto the method described in J. Biol. Chem. 149, 413 (1943)) was dissolvedin 600 ml. of hot 0.3 N hydrochloric acid and the solution was boiledunder .3

The precipitate which anol and the solution was treated with 30 ml. of amixture of onev part triethylamine: one part acetic acid: five partsmethanol, thereby converting the hydrochloride of the ACTH hydrolysateto the corresponding crude free base which was washed with four 15 ml.portions of methanol and dried. The crude free base of the ACTHhydrolysate was dissolved in 75 ml. of the aqueous phase resulting froma mixture of three parts of butanol: two parts of liquefied phenol: fiveparts of water, at which point a small insoluble residue was removed anddiscarded. The aqueous solution of the crude free base of the ACTHhydrolysate was washed with four 75 ml. portions of the organic phase ofthe above-butanolphenol-water mixture, and the organic phase wasdiscarded after each washing.

The aqueous phase was then extracted with one 75 ml. portion ofliquefied phenol thereby transferring the free base of the ACTHhydrolysate to the phenol layer. The phenol solution was diluted with750 ml. of ether and the resulting mixture extracted with three 75 ml.portions of water which had been acidified with hydrochloric acid.

The combined aqueous extracts were washed three times with ether andconcentrated to dryness to produce the hydrochloride of the ACTHhydrolysate in purified form. This material was further purified andfreed of residual HCl by dissolving it in about 20 ml. of methanol andadding 200 ml. of ether. The precipitate which formed was collected,washed with ether, and dried in vacuo to produce 0.76 g. of thehydrochloride of the ACTH hydrolysate in highly purified form. Thisfinal product showed an activity in the rat assay of 630% with respectto Armour Standard ACTH. It showed no antidiuretic activity when testedin animals.

Example 6 One gram of hog crude prolactin, prepared as described in thearticle by Fishman (J. Biol. Chem. 167, 425 (1947) was treated with 20ml. of boiling 0.3 N hydroc'hloric acid, and the mixture was boiled forone hour, cooled, and after the addition of two drops of tn'butylcitrate to prevent foaming it was concentrated to dryness in vacuo. Theproduct, a grayish purple solid, was triturated with three 5 ml.portions of methanol. The methanol-insoluble residue was discarded, andthe combined clear methanolic extracts were treated with 150 ml. ofether, giving a light-colored precipitate. The preciptiate was collectedby centrifugation and dried in vacuo. The product weighed 547 mg. andshowed an ACTH assay value of 120%. It was tested in animals and foundto be devoid of antidiuretic activity.

The product was tested clinically in a patient with rheumatoidarthritis. It was given for four days, 81 mg. the first day, and between37 and 48 mg. for the next three days. It produced an excellentremission of the arthritis, with no significant sodium retention orantidiuretic effect. The patients sedimentation rate droppedsatisfactorily.

Example 7 To 1.0 g. of hog crude prolactin (the same starting materialutilized in Example 6 hereinabove) was added 8.5 ml. of 6 N hydrochloricacid, and the mixture (pH below 0) was kept at 37 C. for four hours. Itwas then diluted to ml. with water and lyophilized.

The resulting white solid was triturated with three 5 ml. portions ofmethanol. The methanol-insoluble material was discarded and the combinedmethanol extracts treated with 150 ml. of ether. The resulting whiteprecipitate was collected and dried. It weighed 386 mg., and showed ananimal ACTH assay value of However, animal tests showed the presence inthe sample of antidiuretic material.

Example 8 A 60 g. portion of ACTH concentrate (hog acidacetone powder)was treated with 1200 m1. of hot 0.3 N hydrochloric acid and thesolution (ph about 0.5), was heated under reflux for one hour, cooled,and after addition of three drops of tributyl citrate it wasconcentrated to dryness in vacuo. The wet residue was dried bylyophilization. The product was extracted with 600 ml. of methanol andthe methanol solution, after removal of the insoluble portion, waspoured into ca. 3.5 1. of ether. The resulting precipitate wascollected, Washed with 600 ml. of ether, and dried. The product weighed27 g., and had an ACTH activity of 120%. It was non-toxic, and showed nosign of antidiuretic effect.

Example 9 A 1.77 g. portion of crude prolactin obtained from beefpituitary glands was hydrolyzed by dissolving in 25 ml. of 0.3 Nhydrochloric acid and heating the solution (pH about 0.5) under refluxfor one hour. The solvent was removed by lyophilization and the'driedproduct extracted with 12 ml. of methanol. An insoluble fraction wasremoved and the methanol solution was poured into 100 ml. of ether. Theresulting precipitate was collected, washed with 20 ml. of ether, anddried in vacuo. The product, 555 mg, had an assay value of 80%.

After further purification of this material utilizing the proceduredescribed in Example hereinabove, there was obtained a purified ACTHhydrolysate which was free of antidiuretic efiect, and which had anassay value of Example 10 A 1.279 g. portion of crude prolactin obtainedfrom sheep pituitary glands was hydrolyzed by heating for one hour in0.3 N hydrochloric acid at 100 C. The solution was lyophilized and theproduct extracted with 17 ml. of methanol. The methanol solution wastreated with 100 ml. of ether. The resulting precipitate was washed with20 ml. of ether and dried in vacuo. It weighed 632 mg, and had an assayvalue of 85%.

After further purification of this material utilizing the proceduredescribed in Example 5 hereinabove, there was obtained a purified ACTHhydrolysate which was free of antidiuretic elfect, and which had anassay value of 560%.

Example 11 Ten grams of an ACTH concentrate of activity 190% wasprepared by extracting defatted hog pituitary glands with acetic acidand precipitation of the material from the acetic acid solution bydilution with ether (cf. Astwood, Cleroux, Payne, and Raben, I. New Eng.Med. Center, January 1950, p. 2). The material was hydrolyzed bydissolving it in 200 ml. of 0.3 N hydrochloric acid, and refluxing thesolution for one hour. The solution (pH about 0.5) was cooled,concentrated, and the wet residue dried by lyophilization. The productwas extracted with 100 m1. of methanol, an insoluble portion removed,and themethanol solution was poured into 1 liter of ether. The resultingprecipitate was collected, washed with 200 ml. of ether, and dried. Itweighed 2.74 g. and had an activity of 170%. It had no antidiureticaction.

In a modification of the procedure described above, the lyophilizedhydrolysate was extracted with 90% acetic acid-10% water instead ofmethanol, and the acetic acid extract (200 ml.) was poured into 2 l. ofether. A product similar to that described above was obtained, but theyield was increased to 6.51 g.

The above procedure was repeated on a corresponding ether precipitate ofan acetic acid extract of sheep pituitary glands, using the methanolextraction of the lyophilized hydrolysate. From 20 g. of material, 2.34g. of product, with an activity of 110% and free of antirliureticmaterial, was obtained.

10 Example 12 A 100 mg. portion of hog acid-acetone powder was dissolvedin 5 ml. of 0.3 N sulfuric acid, and the solution (pHabout 0.5) washeated under reflux for one hour. It was then adjusted to pH 3.9 byaddition of barium hydroxide solution. The precipitate was removed, andthe clear supernatant solution lyophilized. The dried residue wasextracted with methanol, and the methanol solution treated with 45 ml.of ether. The resulting precipitate was washed with ether and dried. Ithad an activity of 105%, and showed no antidiuretic action.

Example 13 activity of 110%, and showed no antidiuretic action.

Example 14 A g. portion of ACTH concentrate (hog acidacetone powder) wastreated with 1200 m1. of hot 0.3 N hydrochloric acid and the solutionwas heated under reflux for one hour, cooled, and after addition ofthree drops of tributyl citrate it was concentrated to dryness in vacuo.The wet residue was dried by lyophilization. The .product was extractedwith 600 ml. of methanol, and the methanol solution, after removal ofthe insoluble portion, was poured into ca. 3.5 l. of ether. Theresulting precipitate was collected, washed with 600 ml. of ether, anddried.

This product was extracted with 300 ml. of methanol, and the insolubleresidue discarded. To the methanol solution was added 60 ml. of amixture of 1 volume of triethylarnine: 1 volume of acetic acid: 5volumes of methanol. The resulting free base precipitate was collected,washed with four 30-m1. portions of cold meth-.

anol and then with one 50 ml. portion of ether, after which it was driedin vacuo.

The free base so obtained was dissolved in 150 m1. of the aqueous phaseresulting from the mixture of three volumes of butanol: two volumes ofliquefied phenol (88%): five volumes of water. This aqueous solution wasthen washed with four 150 m1. volumes of the organic phase from theabove mixture. After the washing, the aqueous phase was extracted with150 ml. of liquefied phenol. The liquefied phenol extract was dilutedwith about 1500 ml. of ether and extracted with three 150 ml.

portions of water weakly acidified with hydrochloric acid. The combinedacidic aqueous extracts were washed with three l-l. portions of etherand then concentrated to dryness. The product was dissolved in 30-40 ml.of methanol and poured into ca. 350 ml. of ether. The precipirate wascollected, washed with ml. of ether, and dried in vacuo 1E0 give 1.95 g.of purified ACTH hydrolysate having an assay value of 725% Example 15 anadrenocorticotropic activity approximately ten times that of ArmoursStandard ACTH LaJ-A, was dissolved, in 10 ml. of 0.3 N aqueoushydrochloric acid and the solution (pH about 0.5) was boiled underreflux for a 1 1 period of 45 minutes. The solution (pH about 0.5) wascooled and evaporated from the frozen state. The residual material wasdissolved in methanol and precipitated therefrom by the addition ofether. The precipitated material was recovered by filtration and driedto give 63 mg. of hydrolysate of said ACTH pepsin digest.

In the rat assay, the product was found to possess ACTH activity equalto ten times the activity of Armour Standard ACTH. The therapeutic doseof standard ACTH is of the order of 1.2 mg/kg; the therapeutic dose ofthis ACTH hydrolysate is therefore approximately 0.12 ing/kg. When thisACTH hydrolysate was tested using the rat test described in Example -1hereina'bove, it showed no antidiuretic properties when administered tothe animals at a dose level of 0.12 mg./ kg.

Example 16 Sixty grams of porcine acid-acetone powder (prepared asdescribed by Li et al., J. Biol. Chem. 149, 41-3 (1943) for sheepacid-acetone powder) was dissolved in 600 ml. of boiling 0.3 Nhydrochloric acid and the solution was boiled under reflux for a periodof one hour. The resulting solution (pH about 0.5) was cooled, andevaporated to dryness in vacuo, below room temperature. The residualmaterial was triturated with 600 ml. of methanol, the methanol-insolubleresidue was discarded, and the methanolic solution was poured into 6liters of ether, whereupon a light-colored precipitate was formed. Theprecipitate was recovered by centrifugation, washed with ether, anddried in vacuo to give an ACTH hydrolysate which showed an assay valueof 120% and no antidiuretic activity in the rat test.

The product was tested clinically in a patient with rheumatoidarthritis. The patient was a 49 year old white male with a twenty-yearhistory of typical rheumatoid arthritis. At the time of this treatment,he had advanced changes involving all peripheral joints. There werecontractures, deformities, limitation of motion, morning stiffness, anddiffuse rest pain. He was confined to bed in the supine position.

Multiple doses of Armour ACTH in this patient had in the past causeddefinite but not dramatic improvement,

irreparable destructive changes having been blamed for the lack ofdramatic response. Moreover, objective evidence of improvement hadalways been obtainable in the form of a fall of the erythrocytesedimentation rate.

The patient was observed in the present study for a period of seventeendays. He received no medication during the first eight and last two daysof the period. During days 915, he received the ACTH hydrolysatedescribed above in doses of 12.5 mg. every six hours. Foradministration, the sample was dissolved in saline, sterilized byfiltration through a Seitz filter, and given by intramuscular injection.

On the morning of day 10 (one day after start of treatment) there was adefinite decrease in morning stiffness, together with increased agility.The patientreported that his sleep had been uninterrupted for the firsttime in weeks. By day 15, he was much better. He slept well, and wasable to sit up on the edge of the bed.

The patients erythrocyte sedimentation rate, which had been between 38and 43 mm. (Westergran method) during the pre-treatment period, fell to19-23 mm. during the last three days of treatment (a nearly normalvalue). No retention of water could be detected, nor was a significantdeviation from the normal rate of sodium excretion noted.

Various changes-and modifications may be made in carrying out thepresent invention, without departing from the spirit and scope thereof.Insofar as these changes and modifications are within the purview of theannexed claims, they are to be considered as part of our invention.

We claim:

1. The process of converting an ACTH substance, derived from pituitaryglands and characterized as having 12 both adrenocorticotropic andantidiuretic activity, to an ACTH hydrolysate having substantiallyunimpaired adrenocorticotropic activity and substantially noantidiuretic activity, which comprises heating a solution of said ACTHsubstance in an aqueous non-oxidizing mineral acid, the normality ofsaid solution being between about 0.2 N and 0.6 N throughout the heatingperiod, at a temperature be tween about 60 and 115 C., for a period oftime varying from a minimum of about fifteen minutes when utilizinghydrolysis conditions within the range 0.3 N acid at about lO0-115 C. to0.6 N acid at about 8-1 C., to a maximum of about two hours whenutilizing hydrolysis conditions within the range 0.2 N acid at about-115 C. to 0.6 N acid at about 6080 C.

2. The process of converting an ACTH substance, derived from pituitaryglands and characterized as having both adrenocorticotropic andantidiuretic activity, to an ACTH hydrolysate having substantiallyunimpaired adrenocorticotropic activity and substantially noantidiuretic activity, which comprises heating a solution of said ACTHsubstance in an aqueous non-oxidizing mineral acid under reflux at atemperature of about 100 C. for a period of time within the range ofabout thirty minutes to two hours, the normality of said solution beingapproximately 0.3 N throughout the heating period.

3. The process of converting an ACTH substance, derived from pituitaryglands and characterized as having both adrenocorticotropic andantidiuretic activity to an ACTH hydrolysate having substantiallyunimpaired adrenocorticotropic activity and substantially noantidiuretic retic activity, which comprises reacting the ACTH substancewith an aqueous non-oxidizing mineral acid, the normality of thereaction solution throughout the reaction period being within the rangeof approximately 0.2 N to 0.6 N, said reaction being carried out byheating the reactants together at a temperature within the range ofapproximately 60 to C. for a period of time ranging from more than 15minutes to less than four hours sufiicient to substantially eliminatethe antidiuretic activity of the ACTH substance without substantiallyimpairing its adrenocorticotropic activity.

4. The process of claim 3 wherein the non-oxidizing mineral acid ishydrochloric acid.

5. The process of claim 3 wherein the non-oxidizing mineral acid issulfuric acid.

6. The process of claim 3 wherein the non-oxidizing mineral acid ishydrobromic acid.

7. The process of converting an ACTH substance, derived from pituitaryglands and characterized as having both adrenocorticotropic andantidiuretic activity, to an ACTH hydrolysate having substantiallyunimpared adrenocorticotropic activity and substantially no antidiureticactivity, which comprises reacting the ACTH substance with an aqueousnon-oxidizing mineral acid, the normality of the reaction solutionthroughout the reaction period being approximately 0.3 N, and thereaction being carried out by heating the reactants together at 100 C.for a period of one hour.

8. The process of converting an ACTH substance, derived from pituitaryglands and characterized as having both adrenocorticotropic andantidiuretic activity, to an ACTH hydrolysate having substantiallyunimpaired adrenocorticotropic activity and substantially noantidiuretic activity, which comprises heating a solution of said ACTHsubstance in an aqueous non-oxidizing mineral acid at a temperature of100 C. for a period of one hour, the normality of said solutionthroughout the heating period being within the range of about 0.2 N toabout 0.6 N.

9. The process of converting an ACTH substance, derived from pituitaryglands and characterized as having both adrenocorticotropic andantidiuretic activity, to an ACTH hydrolysate having substantiallyunimpaired adrenocorticotropic activity and substantially noantidiuretic activity, which comprises heating a 0.3 N aqueousnonoxidizing mineral acid solution of said ACTH substance at 100 C. fora period of time ranging from more than 13 15 minutes to less than 4hours suflicient to substantially eliminate the antidiuretic activity ofthe ACTH substance.

10. The process of converting an ACTH substance, derived from pituitaryglands and characterized as having both adrenocorticotropic andantidiuretic activity, to an ACTH hydrolysate having substantiallyunimpaired adrenocorticotropic activity and substantially noantidiuretic activity, which comprises heating a 0.3 N aqueousnonoxidizing mineral acid solution of said ACTH substance at atemperature within the range of more than 60 C. to less than 115 C. fora period of time suflicient to substantially eliminate the antidiureticactivity of the ACTH substance which time is approximately one hour.

11. The process of converting an ACTH substance derived from pituitaryglands and characterized as having both adrenocorticotropic andantidiuretic activity, to an ACTH hydrolysate having substantiallyunimpaired adrenocorticotropic activity and substantially noantidiuretic activity, which comprises heating a solution of said ACTHsubstance in an aqueous non-oxidizing mineral acid at a temperatureWithin the range of approximately 60 to 115 C. for a period of time tosubstantially eliminate the antidiuretic activity of the ACTH substanceranging from 30 minutes to 1 hour, the normality of said solutionthroughout the heating period being within the range of approximately0.2 N to 0.6 N.

12. The process of converting an ACTH substance, derived from pituitaryglands and characterized as having both adrenocorticotropic andantidiuretic activity, to an ACTH hydrolysate having substantiallyunimpaired adrenocorticotropic activity and substantially noantidiuretic activity, which comprises heating a 0.2 N aqueousnon-oxidizing mineral acid solution of said ACTH substance at atemperature within the range of approximately 100 to 115 C. and for aperiod of time sufiicient to substantially eliminate the antidiureticactivity of the ACTH substance ranging from 1 to 2 hours.

13. The process of converting an ACTH substance, derived from pituitaryglands and characterized as having both adrenocorticotropic andantidiuretic activity, to an ACTH hydrolysate having substantiallyunimpaired adren-ocorticotropic activity and substantially noantidiuretic activity, which comprises heating a solution of said ACTHsubstance in an aqueous non-oxidizing mineral acid at a temperaturewithin the range of approximately 60 to 80 C. for a period of timesufiicient to substantially eliminate the antidiuretic activity of theACTH substance ranging from 1 to 2 hours, the normality of said solutionbeing approximately 0.6 N throughout the heating period.

14. The process of converting an ACTH substance, derived from pituitaryglands and characterized as having both adrenocorticotropic andantidiuretic activity, to an 14 ACTH hydrolysate having substantiallyunimpaired adrenocorticotropic activity and substantially noantidiuretic activity, which comprises heating a 0.3 N aqueousnon-oxidizing mineral acid solution of said ACTH substance at atemperature within the range of 100 to 115 C. for a period of timesuflicient to substantially eliminate the antidiuretic activity of theACTH substance ranging from over 15 minutes to 1 hour.

15. The process of converting an ACTH substance, derived from pituitaryglands and characterized as having both ad-renocorticotropic andantidiuretic activity, to an ACTH hydrolysate having substantiallyunimpaired adrenocorticotropic activity and substantially noantidiuretic activity, which comprises heating a 0.6 N aqueousnon-oxidizing mineral acid solution of said ACTH substance at atemperature of approximately to C. for a period of time sufiicient tosubstantially eliminate the antidiuretic activity of the ACTH substanceranging from over 15 minutes to 1 hour.

References Cited in the file of this patent UNITED STATES PATENTS1,860,469 Lautenschlager May 31, 1932 2,073,354 Schoeller et al. Mar. 9,1937 2,085,768 Schoeller et al. July 6, 1937 2,337,823 Junkmann Dec. 28,1943 2,362,993 Fleischer et a1 Nov. 21, 1944 2,370,154 Fleischer Feb.27, 1945 FOREIGN PATENTS 328,347 Great Britain May 1, 1930 OTHERREFERENCES Collip: Symposia Quant. Biol., vol. 1937, p. 212.

Li: Macy Foundation, Transactions of the 17th Meeting, Conf. onMetabolic Aspects of Convalescence, pp. 114-121, 1948.

Recent-Progress in Hormone Res, vol. 7, 1952, pp. 59-73, p. 73 esp.pert, Academic Press Inc., N. Y. C.

Li et al.: Article in J. Biol. Chem, vol. 149, August 1943, pp. 413-424.

Tyslowitz: Article in Science, September 3, 1943, pp. 225-226.

J. A. M. A., April 29, 1950, p. 1366.

I. A. C. 5., June 1950, pp. 2815-2816.

Geschwind et al.: Articles in Science," October 13, 1950, vOl. 112, PP.436-437.

Payne et al.: Article in J. Biol. Chem., December 1950, pp. 719-731.

Reinhart et al.: Proc. Soc. Exptl. Biol. and Med, April 1951, pp.836-838.

Pincus: The Hormones, 1948, Academic Press Inc., N. Y. C., vol. 1, pp.667-671 and vol. 2, p. 469.

1. THE PROCESS OF CONVERTING AN ACTH SUBSTANCE, DERIVED FROM PITUITARYGLANDS AND CHARACTERIZED AS HAVING BOTH ADRENOCORTICORTROPIC ANDANTIDIURETIC ACTIVITY, TO AN ACTH HYDROYLSATE HAVING SUBSTANTIALLYUNIMPAIRED ADRENOCORTHICOTROPIC ACTIVITY AND SUBSTANTIALLY NOANTIDIURETIC ACTIVITY, WHICH COMPRISES HEATING A SOLAUTION OF SAID ACTHSUBSTANCE IN AN AQUEOUS NON-OXIDIZING MINERAL ACID, THE NORMALITY OFSAID SOLUTION BEING BETWEEN ABOUT 0 2 N AND 0.6 N THROUGHOUT THE HEATINGPERIOD, AT A TEMPERATURE BETWEEN ABOUT 60 TO 115*C., FOR A PERIOD OFTIME VARYING FROM A MINIMUM OF ABOUT FIFTEEN MINUTES WHEN UTILIZINGHYDROLYSIS CONDITIONS WITHIN THE RANGE 0.3 N ACID AT ABOUT 100-115*C. TO0.6 N ACID AT ABOUT 8-1*C., TO A MAXIMUM OF ABOUT TWO HOURS WHENUTILIZING HYDROLYSIS CONDITIONS WITHIN THE RANGE 0.2 N ACID AT ABOUT100-115*C. TO 0.6 N ACID AT A BOUT 60-80*C.